This invention deals with the preparation of salts of acyl phosphoric acids, more particularly with the preparation of ammonium salts of acyl phosphates, such as diammonium acetyl phosphate. Such compounds are useful in the production of phosphorylating agents such as adenosine triphosphate (ATP), a required cofactor in many reactions in the biosynthesis of materials such as protein, carbohydrates, nucleotides, nucleic acids, and terpenes. The products of the present invention can also be used as phosphorylating agents themselves or as acylating agents, e.g. diammonium acetyl phosphate reacts with aniline to form acetanilide in aqueous media at room temperature.
Biosynthesis by enzymatic catalysis has been attracting increased attention as a means of large scale production of many complex products. See, e.g., Skinner, "Enzymes Technology," Chem. & Engin. News, Vol. 53, No. 33, p. 22 (Aug. 18, 1975). A major limitation on the commercial usefulness of such processes, however, has been the cost of many of the cofactors or coenzymes necessary to conduct the reactions involved. For example, ATP is a cofactor which plays a prominent role in many biosynthetic processes, promoting formation of chemical bonds which otherwise would not form in significant quantities in dilute aqueous solutions. See generally Stadtman, "The Enzymes", Vol. 8, chapter 1 (3rd ed. 1972). For the use of ATP in cell-free enzymatic synthesis of the cyclic decapeptide antibiotic Gramicidin S, see Gardner et al., Enzyme Engineering 2:209 (1974); Whitesides et al., Enzyme Engineering 2:217 (1974); and Hamilton et al., Enzyme Engineering 2:133 (1974), all incorporated herein by reference. The cost of cofactors such as ATP is extremely high (reported by Skinner, supra, at $67 per gram), which has severely retarded the use of such processes on a commercial scale.
In order to reduce the cost of enzymatic synthesis requiring ATP, a system has been developed by enzymatic regeneration of ATP from adenosine diphosphate (ADP) and/or adenosine monophosphate (AMP). Normally treatment of the raw materials used in biosynthesis with ATP to form more complex products results in the consumption of ATP and the production of AMP and/or ADP. Under the regeneration system, if AMP is produced in the biosynthesis, it is converted to ADP by enzyme catalyzed phosphoryl transfer from ATP according to the following equation: ##EQU1## The ADP is then converted to ATP by reaction with a phosphate donor, e.g. acetyl phosphate (AcP), catalyzed by a phosphotransferase enzyme, e.g. acetate kinase: ##EQU2## Whitesides et al., supra, and Gardner et al., supra, both discuss this regeneration scheme. While this significantly reduced the difficulty in obtaining ATP, it did not eradicate it, largely because commercially acceptable methods of producing the phosphate donors such as acetyl phosphate were not known.
Acetyl phosphate had previously been synthesized from phosphoric acid by acylation with various ingredients, including acetyl chloride, ketene, isopropenyl acetate, and acetic anhydide, followed by isolation as the lithium or silver salts. See Whitesides et al., "Large-Scale Synthesis of Diammonium Acetyl Phosphate," J. Org. Chem., 40:2516 (1975) and references cited, incorporated herein by reference. All of these procedures involve difficult work-up and isolation sequences, and none of them are suitable for the preparation of acetyl phosphate in large quantity.
The previously known processes of producing acetyl phosphate by acylation of phosphoric acid with ketone are discussed in Whitesides et al., supra, and in Bently, "A New Synthesis of Acetyl Dihydrogen Phosphate," J. Amer. Chem. Soc. 70:2183 (1948). See also Kasalopoff et al., "Organic Phosphorous Compounds," Vol. 6:294, at 295-6 (1973). Generally, ketene, obtained for example from the cracking of acetic acid or acetone, was reacted with 85% phosphoric acid dissolved in ether to produce a mixture of monoacetyl, diacetyl and triacetyl phosphates, depending on factors such as concentrations of the reactants and the length of reaction, together with other reaction products. The acetyl phosphates were then isolated either as the silver salts or as the lithium salts. Isolation as the silver salt involved an ice water wash, precipitation of unreacted phosphate with barium hydroxide, and treatment of the remaining barium acetyl phosphate solution with excess ice-cold silver nitrate. The disilver acetyl phosphate would then be converted into acetyl phosphate by treatment with hydrogen sulfide in aqueous solution, or by suspension in ether and treatment with ethereal hydrogen chloride. The disilver acetyl phosphate could also be converted to diammonium acetyl phosphate, first by converting the silver salt to the free acid in ether, followed by reaction with dry ethereal ammonia. See Bently, supra, at 2183-4. Isolation as the dilithium salt involved neutralization with aqueous lithium acetate, carbonate or hydroxide. Dilithium acetyl phosphate was then precipitated by addition of ethanol. It was then necessary to remove water from the dilithium acetyl phosphate without hydrolyzing it. Following this procedure acyl phosphate was obtained in yields of about 50%, based on the starting phosphoric acid.
Clearly, these previous procedures for preparing usable acetyl phosphate were very difficult as best. They require a neutralization step of the acetyl phosphoric acid in aqueous solutions, followed by the use of very expensive silver or lithium salts to effect isolation of the acetyl phosphate. Neutralization with the lithium salts, e.g. lithium acetate, carbonate or hydroxide, results in phosphate "slimes", through which it is difficult to separate the mother liquor. Removal of water from dilithium acetyl phosphate without hydrolyzing it required a time consuming and not always successful lyophilization or related procedure. Yields were relatively low, and the silver salt had to be converted to the sodium, potassium, or other salt before it could be used to regenerate ATP.
Accordingly, it is an object of the present invention to provide a simple, direct method of producing acyl phosphates. It is a further object to provide a simple, inexpensive method of producing phosphate donors suitable for use in biosynthesis. It is a more particular object to provide a method of producing phosphate donors useful in production of ATP. It is a further object to provide a process for making ATP utilizing ammonium acyl phosphates, more particularly diammonium acetyl phosphate. It is a further object of this invention to provide a process of preparing acyl phosphates in high yield, without the need for expensive lithium or silver precipitation. It is a still further object to provide a method of preparing acyl phosphate salts which are highly stable, and can be used directly as a phosphate donor, without having to be converted to dihydrogen acyl phosphate, more particularly, the ammonium acyl phosphate salts. It is a still further object to provide a method of preparing acetyl phosphate salts, more particularly diammonium acetyl phosphate, which is efficient and economical enough for use on an industrial scale.
Further objects of the invention will be apparent to those skilled in the art from a consideration of the present disclosure, and/or from practice of the invention disclosed herein.